Anion exchangers (AE) constitute a diverse family of mammalian plasma membrane transport proteins which contribute to the regulation of intracellular and extracellular ionic homeostasis and in erythroid cells, to the stability of the plasma membrane. Understanding the structure, function and regulation of this nearly ubiquitous class of transporter holds promise for understanding the molecular pathogenesis of such diverse disorders as anemia, osteoporosis, ulcer, renal failure and post-trauma cerebral edema. The research described in this proposal is focused on elucidating the molecular mechanism of AE function. Five specific aims are proposed. (1) To define the functional topology of the membrane domain of AE1. We will test the hypothesis that functionally important components of the anion translocation pathway are contained within non-alpha-helical segments that are not predicted to be transmembrane. To this end we will combine site-directed mutagenesis and covalent cysteine chemistry to produce a topological map of the membrane domain of AE1. (2) To determine the molecular mechanisms of AE2/AE3 regulation. We will test the hypothesis that interaction of the two domains of AE is required for regulation by internal protons. To this end we propose to identify interacting determinants on both domains that contribute to the regulation of AE activity by intracellular protons, divalent cations and phosphorylation. (3) To identify a cytoskeletal or cytosolic ligand for AE2/AE3. We will test the hypothesis that the N-terminal cytoplasmic domains of AE2/AE3 interact with an ankyrin-like protein or other cytosolic ligand. To this end we propose to determine the binding affinity of these anion exchangers to newly identified ankyrins and to use a yeast 2-hybrid screen to identify other potential interacting proteins. (4) To determine the mechanism by which AE1 stabilizes the RBC plasma membrane. We will test the hypothesis that the severe anemic phenotype of the AE1 ~knochout~ mouse is due to the combined loss of multiple AE1 functions. We will rescue some of these phenotypes by replacement of AE1 with partial function mutants and chimeric proteins. (5) To determine the role of ankyrin binding in AE1 biosynthesis. We will test the hypothesis that the stability and targeting of AE1 to the plasma membrane depends on its association with ankyrin early in the biosynthetic pathway.